a. Field Of The Invention
The present invention is directed to a stabilized internal calibrator for use on a membrane-based test device, particularly a diagnostic test device as used in human and veterinary medicine. More particularly, the present invention is directed to a covalently cross-linked antigen-antibody complex for use as a stabilized internal calibrator on a calibration zone of any membrane-based device. In the present invention, the stabilized internal calibrator is more resistant to degradative processes, such as oxidation, enzymatic digestion and leaching, and requires less components and steps than conventional internal calibration systems.
b. Background
Membrane-based test devices, particularly those devices used in diagnostic and veterinary medicine, employ a variety of internal and external calibrators to provide a qualitative or a quantitative result for an analyte of interest in a test solution. One type of membrane-based test device is the dipstick. The dipstick is a stick having a small reagent impregnated membrane at one end for dipping into a test solution either containing or suspected of containing the analyte of interest. The dipstick membrane (hereinafter "dipstick") develops a color that is proportional to the concentration of the analyte of interest in the test sample. Typically, the user determines the concentration of the analyte by comparing the color on the membrane to the color on an external calibrator, such as a series of colored plates that are printed on a label. The color of each plate is associated with a particular concentration of the analyte. The color on the plate that most closely approximates the color on the dipstick provides the user with an approximate concentration of the analyte in the test sample.
External calibration of a dipstick, via colored plates, has several problems. First, it is difficult to match the color of the plates with the color on the dipstick. Secondly, the color on the plates would not fade in proportion to the adverse conditions affecting the color on the dipstick. Further, the color on the plates would at best only be accurate for a particular set of reaction conditions. However, in the normal clinical situation, the dipstick reaction is often performed at other than the calibration conditions.
Accordingly, it is an object of the present invention to provide a stabilized internal calibrator for a dipstick that would compensate for conditions affecting the test reagents prior to and during their reaction with the analyte of interest.
More recently, the color produced by a dipstick that has been dipped into a test sample is read on an instrument. Prior to reading the color produced by a test sample, the instrument is calibrated against the color produced by a second dipstick that has been exposed to a known concentration of analyte. The dipstick method suffers from several problems. The first problem with this dipstick method is that it requires two dipsticks. Secondly, the instrument may have drifted from the values set in its last calibration, which may have only been hours before. Further, the reaction conditions between the calibrator and the test sample may have been different, either with respect to time or reaction temperature. Accordingly, it is an object of the present invention to eliminate the need for two dipsticks to calibrate a dipstick reaction. It is a further object of the present invention to insure that the calibrator and test sample are exposed to the same reaction conditions.
A second type of membrane-based test device is the ICON.RTM. type device of Hybritech's U.S. Pat. Nos. 4,632,901, 4,727,019 and 5,120,504. Although the word Immunoconcentration.TM. (also known as ICON.RTM.) is a trademark of Hybritech Incorporated, other manufacturers of a similarly functioning devices, such as Pacific Biotech, Inc., refer to their device as an immunosorbent assay. In either of these devices, an excess amount of the binding partner to the analyte of interest is immobilized, typically as a test spot, on a membrane that is positioned over an absorbent pad. The pad is in liquid receiving relationship with the membrane. When an aliquot of a test sample that is suspected of containing the analyte of interest is placed on the membrane, it is wicked through the membrane and into the absorbent pad. Any analyte of interest that passes through the test spot on the membrane is bound to its binding partner immobilized thereon. A reagent containing a labeled second binding partner to the analyte is poured onto the membrane. As it passes through the membrane, the labeled binding partner to the analyte of interest binds to any immobilized analyte at the test spot thereon in proportion to the amount of analyte that has been immobilized. Ultimately, a color is produced at the test spot on the membrane in proportion to the amount of analyte immobilized thereon.
In the ICON.RTM. type devices, it is desirable to provide the user with an internal reference, such as a calibration or reference spot, that produces a visible colored signal in proportion to a known concentration of an analyte of interest. This internal reference allows the user of the ICON.RTM. type device to obtain a semi-quantitative result (i.e., a result reported as greater than, equal to, or less than the calibrator). To obtain the semi-quantitative result, the user of the ICON.RTM. type device visually compares the color produced at the test spot relative to the color produced at the calibration spot and determines whether it is greater than, equal to, or less than the color produced by the known concentration of the calibrator at the calibration spot. Alternatively, the user of the device can quantitate the concentration of the analyte of interest by instrumentally measuring the color of the known reference and the color of the unknown sample, such as by reflectance, and solving an equation for the concentration of analyte in the unknown.
One method for providing an internal reference in an ICON.RTM. type device involves binding (i.e., immobilizing) a second antibody, typically in the shape of a spot, to a discrete portion of the membrane. Alternatively, the second antibody may be immobilized on a plurality of microparticles that themselves have been immobilized, such as by trapping or bonding, to a discrete spot on the membrane's surface. See Anderson et al., "Internally Referenced Immuno-Concentration.TM. Assays," Clin. Chem., 32 1692-1695 (1986). Unlike the first antibody at the test spot, the second antibody at the reference spot is not specific for the analyte of interest. Rather, it is specific for an exogenous substance that is selectively foreign to the test samples suspected of containing the analyte of interest. The second antibody is bound to a sufficient amount of the exogenous substance such that when the exogenous substance is labeled, the reference spot produces a detectable signal at a level that corresponds with the signal produced by a known amount of the analyte of interest that has become immobilized and labeled at the test spot under those reaction conditions.
The above method of providing an internal reference using an exogenous substance is laborious due to the necessary cross referencing between the parallel series of reactions involving the analyte and the exogenous substance.
It is also an object of the present invention to eliminate the manufacturing difficulties ( i.e., laboriousness) associated with having a different reaction series for the reference and the analyte of interest.
Attempts to utilize the same immobilized antibody on both the test spot and the reference spot have most often failed. Antibodies have two binding sites. When a known amount of the analyte of interest is bound to the antibody on the reference spot, there is no assurance that all of the antibody binding sites on the reference antibody have been occupied. Further, the desirable signal level for the reference spot may require a less than saturating amount of antibody. Accordingly, when a test solution containing analyte of interest is added to the test device, the analyte of interest is capable of binding both to the antibody on the test spot and to the available sites on the identical antibody on the reference spot. As a result, the amount of analyte that is bound to the antibody at the reference spot is influenced by the amount of analyte that is in the test sample. Thus, any signal produced at the reference spot would be upwardly biased as a function of the amount of analyte in the test sample. Consequently, highly elevated concentrations of analyte in a test sample would be reported artificially low relative to their actual values. Accordingly, it is an object of the present invention to produce an internal calibrator for an immunosorbent device that would be stable under differing reaction conditions but that would not be influenced by the amount of analyte in a test solution.
A third type of membrane-based test device that also has applications in diagnostic and veterinary medicine is the immunochromatography test device. Immunochromatography test devices of various types have been known to the art for years. In test devices of the immunochromatography type, a sample that is suspected of containing the analyte of interest is placed at or near one end of a membrane strip. The sample is allowed to be carried to the opposite end of the membrane strip by a liquid phase that traverses the membrane strip by capillary action. While traversing the membrane strip, the analyte in the test sample, if any, encounters one or more reagents with which it may react to produce a detectable signal. The early types of immunochromatography devices, such as taught in U.S. Pat. No. 4,366,241 (Tom et al.), lacked an internal reference. Later devices, such as taught in U.S. Pat. No. 4,374,925 (Litman) employed an internal reference. In some instances, the internal reference was analogous to that described for the ICON.RTM. devices and suffered from the same laborious cross-referencing. In other instances, the internal reference served the function of merely validating a positive signal at the reaction zone, such as by capturing a labeled antibody at a point beyond the reaction zone.
Thus, it is a further object of the present invention to provide an internal calibrator for use with an immunochromatography device that solves the problems in common with the immunosorbent type devices and/or that renders the immunochromatography device capable of approximating the amount of an analyte of interest in a test sample. It is also an object of the present invention to provide an internal calibration zone on a membrane-based device that reflects the stability of the reagents thereon and that is capable of providing a stable calibration signal for calculating the concentration of an analyte of interest in the test sample.